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    Fluorescence spectroscopy becoming key to identifying pain

Fluorescence spectroscopy becoming key to identifying pain

Mikhail Berezin’s focus wasn’t always pain. He was interested in optics.

He liked how molecules interacted with light, and measuring these interactions. He also enjoyed designing and making materials that were light active, specific to different light stimulus.

Then a member of his family began chemotherapy. That’s when his research turned to pain.

Berezin, a Ph.D., is the director of the Optical Spectroscopy Core Facility at Washington University. He is an associate professor of radiology at the School of Medicine and an affiliated professor of chemistry in the Department of Radiology.

Pain

Inflammation causes pain because the swelling pushes against sensitive nerve endings.

Most of pain that we experience comes from the inflammation of our peripheral nerves. The peripheral nerves travel to various parts of our body, carrying signals to and from the brain.

His family member began experiencing chemotherapy induced peripheral neuropathy – something with which Berezin wasn’t familiar. Chemotherapy can cause damage to the peripheral nerves. Symptoms include tingling, burning, numbness, pain, and difficulty with fine motor skills.

So Berezin turned his focus to imaging inflammation in the body – and how to locate it. That, he hoped, would eventually lead to the treatment of peripheral neuropathy and chemotherapy induced peripheral neuropathy in particular.

“I changed the direction of what my lab was doing,” he said. “To do imaging and diagnostics and understand what chemotherapy induced peripheral neuropathy is about.”

Detecting inflammation with fluorescence

The first step was to locate where the inflammation occurred.

Damaged nerves result in the activation of the inflammatory cells that produce reactive oxygen species. Immune cells produce most of the reactive oxygen species.

“We developed probes that detect inflammation based on that,” he said.

The probes are organic molecules that become fluorescent in the presence of inflammation. These work by inducing the fluorescence when the probes are in contact with the reactive oxygen species.

Measuring inflammation

Berezin’s team inject mice with chemotherapy drugs over several months, much like with humans.

The first question was how to measure pain, especially chronic pain, in animals. If he discovered a marker, he would have a parameter to measure. Then he could start treating it with drugs and gauge the results.

“We observed the mice,” he said. “We look at how they behaved. Normally they're on the running wheels, performing a motor activity. If they have chemotherapy drugs, they stop running or slow down until they recover. They also lose appetite. Sometimes they lose sleep.”

But mice can’t tell you when it feels pain. So researchers have to use other methods.

Berezin measured nerve conduction velocity, the speed the signal propagates through the nerve. It’s the time between the introduction of a stimulus and the time when the electrical signal known as action potential reaches the detector.

And it’s a way to identify nerve damage.

“If you touch your finger with a needle, you will feel pain almost immediately,” he said. “But there’s a delay of about 0.1 seconds. If any of the nerves are not okay, it becomes longer. Your sensitivity is getting worse.”

Berezin also looks at bloodwork for markers of pain. He tries to determine what markers appear as the nerves become inflamed. Those markers include chemicals like TNF alpha and proinflammatory cytokines. These are part of the immune response. The body releases these when there is a problem with the nerves.

 “Each organ has specific markers,” he said. “So if your finger is inflamed, that's one set of markers. If you liver is inflamed, it's a different set of markers. And your nerves are probably a third one. There’s a lot of overlap. We're analyzing blood of mice and nerves and looking for those markers.”

Researchers use mice since their nervous system is almost identical to that of humans.

Spectroscopy

working with a highly customized HORIBA Fluorolog modular spectrofluorometer

Helena Hurbon, manager of the Optical Spectroscopy Core Facility and Washington University student working with a highly customized HORIBA Fluorolog modular spectrofluorometer

So how does spectroscopy come into play?

Berezin uses all sorts of optical fluorescence spectroscopy measurements, including fluorescence polarization, lifetime, phosphorescence, and other measurements, to observe the probes exposed to a light source, and to develop new probes. These probes need to be non-fluorescent at the initial introduction into the body and highly fluorescent when the marker is stimulated with light.

If there is an inflammation, it will start producing strong radicals that activate the fluorescence.

“Before you put (the light sources) in contact with reactive oxygen species, you don't see them fluoresce,” he said. “If you dumped only a little bit of reactive oxygen species, you see that they become highly fluorescent.”

The lab

Washington University’s Optical Spectroscopy Core Facility has top-of-the-line spectroscopy and imaging instruments to serve scientists around the globe with the development of optical probes.

At the heart of the lab is a highly modified HORIBA Fluorolog modular spectrofluorometer.  That was the first instrument in the Core purchased from the NIH Shared Instrumentation grant in 2011.

The Optical Spectroscopy Core Facility’s instrument is a monster truck of a spectrometer, dedicated for the near infrared (NIR) and shortwave-infrared (SWIR) spectral ranges. It has three critical parts, measuring fluorescence, fluorescence lifetime, and imaging.

A highly modified HORIBA Flurolog modular spectrofluorometer at Washington University

A highly modified HORIBA Flurolog modular spectrofluorometer at Washington University

Benefits of spectroscopy

The advantage of localizing inflammation has to do with treating particular spots in the body instead of the whole body or symptomatic areas.  For example, with chemotherapy induced peripheral neuropathy, patients often report tingling in the feet and hands, called the gloves and stockings effect. Yet the real inflammation might be somewhere else.

In this syndrome, the problem is not in the distal nerves, as it is with carpal tunnel syndrome. It’s in the dorsal root ganglia, which is next to the spinal cord, because this is where the drugs go.

Fluorescence spectroscopy is also a way to localize tumors that create inflammation that might cause sensations in different parts of the body.

The future

While Berezin experiments with mice, the end game is applying it to human diagnosis and treatment.

And it may come sometime in the near future.

“We are studying some promising markers where we think we can actually start telling people how to treat chemotherapy induced peripheral neuropathy,” he said. “We still need to do more in our mice models to validate our preliminary finding. We will also need to do clinical trials for this, but I think we found an important approach.”

The molecules are known markers for other pain conditions. Which means there are existing drugs to target and treat the pain. But there are hundreds of drugs to treat pain, and none of them worked well for this syndrome.

“We found the one which we believe will work,” he said. “Again, we are about a year ahead, but we might be getting close.”

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